FEBS Letters

Blue light using Flavin (BLUF) proteins are microbial photoreceptors that are involved in various physiological responses. Their occurrence and biochemical properties in fungi remain poorly understood. Here, we investigated a putative BLUF photoreceptor from the corn-smut fungus Mycosarcoma maydis (MmBLUF). Domain analysis, multiple sequence alignment of BLUF core regions, and structural modelling indicated conserved canonical BLUF fold and flavin-pocket residues. However, when heterologously expressed, UV-visible and fluorescence spectroscopy revealed different spectral behaviour than canonical BLUF protein. Further, we tested the role of extended N-terminus in modulation of chromophore binding by expressing N-terminus truncated protein variants. Our results suggest that the unusual spectral behaviour is not linked to the truncation construct (extended N-terminus), which also showed similar spectral features, indicating that the extended N-terminus is unlikely to account for an unusual photodynamics characteristics. Our findings support MmBLUF as a structurally conserved putative fungal BLUF-like photoreceptor with different photochemical properties. Further studies are required to establish its chromophore identity, photocycle and function of this unusual BLUF-like domain from fungal system.

N2-methylguanosine (m2G) is widely found at multiple positions in tRNAs across the three domains of life. Tryptophan tRNA from Thermococcus kodakarensis contains m2G at position 67. We previously proposed that the tRNA m2G methyltransferase Trm14 is responsible for m2G67 formation in tRNATrp from T. kodakarensis, although Trm14 was originally identified as the enzyme catalyzing m2G6 formation in tRNACys in Methanocaldococcus jannaschii. Thus, it remained unclear whether Trm14 could also methylate G67. Here, we characterized archaeal Trm14. Biochemical analyses using recombinant T. kodakarensis Trm14 revealed that the enzyme catalyzes m2G formation at positions 6 and 67 in T. kodakarensis tRNACys and tRNATrp transcripts, respectively. Mass spectrometric analyses demonstrated the loss of m2G6 and m2G67 in native tRNACys and tRNATrp, respectively, from a T. kodakarensis trm14 gene disruptant strain, providing direct evidence for the dual-site specificity of T. kodakarensis Trm14. The growth phenotype of the trm14 gene disruptant strain was comparable to that of the wild-type strain. In contrast, a trm14/trm11 double disruptant, in which trm11 encodes the tRNA m2G10/m22G10 methyltransferase, exhibited severe growth retardation at 95 {degrees}C. This suggests that m2G6/m2G67 and m2G10/m22G10 cooperatively contribute to cellular fitness at high temperatures. Biochemical analyses revealed that Trm14 methylates all 46 T. kodakarensis tRNA transcripts. Furthermore, we found that recombinant M. jannaschii Trm14 methylated both positions. In contrast, the bacterial ortholog TrmN modified only position 6 in tRNA. Overall, this study expands our understanding of archaeal Trm14 by demonstrating its broader substrate specificity and the physiological significance of these modifications under hyperthermophilic conditions.

Ferritins are ubiquitous iron homeostasis proteins found across the tree of life that form conserved 24-subunit cages with octahedral (4-3-2) symmetry. New types of ferritins and ferritin-like proteins are being continuously discovered, such as mini-bacterioferritins, which form smaller shells of 12 subunits, and double-ferritin-fold proteins, which act as ferroxidases but do not form shells. Here, we describe double-ferritin-fold proteins from Asgard archaea, dubbed dFTNs, and determine Cryo-EM structure of a representative from Candidatus Heimdallarchaeum endolithica. The protein forms a dodecameric shell with tetrahedral (2-3) symmetry. N-terminal (NTD) and C-terminal (CTD) domains are bridged by an ordered linker and are related by two-fold rotational pseudosymmetry. C-terminal -helix (helix E) that forms the four-fold channel in classic ferritins is repositioned to be the helix 2 out of 5 ferritin domain -helices in dFTN, with two such helices from NTD and two helices from CTD forming a pseudo-four-fold symmetry structural element. Four three-fold channels are formed by NTDs, and four other such channels are formed by CTDs. The overall arrangement of dFTN ferritin domains is similar to that of protomers in classic ferritin shells. Altogether, our findings expand the range of known ferritin family proteins and provide insight into Asgard archaea iron metabolism.

The type VII secretion system (T7SS) is a membrane-embedded protein export pathway found in mycobacteria and Gram-positive bacteria. Recently it was shown that Mycobacterium abscessus uses its ESX-4 variant of the T7SS to secrete a toxin, EatA, which targets arabinogalactan present in the mycobacterial cell envelope. Prior to its export, EatA forms a complex with a pair of small proteins from the WXG100 family, TapA1 and TapA2. Here we investigated a structural model of the EatA N-terminal domain in complex with TapA1 and TapA2 using site-directed mutagenesis and bacterial 2-hybrid assays. Our results are consistent with the three proteins forming a stacked bundle of alpha-helices. Structural modelling also predicted an interaction of the EatA-TapA1-TapA2 complex with EsxT-EsxU, a second pair of WXG100-family proteins that are likely required for the mechanistic operation of ESX-4. Whilst we could demonstrate a potential interaction between TapA2 and EsxT by bacterial 2-hybrid analysis, we were not able to purify a complex of all five proteins.

BackgroundTuberous Sclerosis Complex (TSC) is a genetic disorder caused by variants in TSC1 or TSC2, leading to mTORC1 hyperactivation and autophagy suppression. Although TSC tumorigenesis typically follows a "two-hit" model, the role of TSC2 haploinsufficiency in autophagy regulation remains unclear. We evaluated autophagy markers in haploinsufficient and gene-edited TSC2 primary cells and investigated the role of metformin in modulating autophagy levels. MethodsPrimary fibroblast cultures were obtained from one healthy individual and three from patients carrying heterozygous germline TSC2 variants: the pathogenic variants c.1008T>G and c.4375C>T.A variant of uncertain significance (VUS) c.724A>T. CRISPR/Cas9-RNP editing was used to model loss of heterozygosity (LOH) in cell pools carrying each variant. Cultures were treated with rapamycin, HBSS, metformin, bafilomycin A1, or vehicle controls, and autophagy was assessed by autolysosomes formation by flow cytometry (acridine orange) and autophagosomes immunofluorescence (LC3 and p-S6K). ResultsIn wild-type cells, only HBSS increased autophagy-positive (acridine orange-positive) cells versus control (15.6% vs. 7.5%; p=0.003). In heterozygous pathogenic cells, rapamycin and metformin increased autophagic cells: c.1008T>G (16.2%, p=0.006; 17.6%, p=0.002) and c.4375C>T (12.5%, p=0.003; 13.3%, p=0.001), versus DMSO controls (9.2% and 7.1%, respectively). VUS c.724A>T cells, with rapamycin increasing autophagic cells (9.74% vs. 6.5%; p=0.0152). In CRISPR-edited cells, all treatments increased the number of autophagic cells compared to the heterozygous cells: c.1008T>G (rapamycin 27.1% vs. 16.7%, p<0.001; metformin 27.2% vs. 17.6%, p<0.001) and c.4375C>T (rapamycin 21.3% vs. 13.1%, p=0.0021; metformin 21.5% vs. 13.6%, p=0.0029). Editing also restored metformin responsiveness in VUS cells (12.5% vs. 8.4%; p=0.0055). Immunochemistry confirmed increased total LC3II and decreased p-S6K across treated cells compared to the control (DMSO). ConclusionThese findings demonstrate that TSC2 haploinsufficiency functionally impairs autophagy prior to second-hit loss. Metformin effectively restores autophagy with phenotypical changes of mTORC1 blockade, highlighting an accessible translational strategy to restore and induce autophagy in TSC cells.

Surface layers (S-layers) are paracrystalline protein lattices that form the outermost layer of the cell envelope in most archaea, providing structural support, protecting against external insults, and co-ordinating interactions with their environment. Despite their widespread occurrence, the molecular and structural details of S-layer architecture in hyperthermophilic archaea remain largely unknown. Here, we report the structure and cellular architecture of the S-layer from the hyperthermophilic archaeon Pyrobaculum arsenaticum by combining in situ electron cryotomography with single-particle electron cryomicroscopy, AlphaFold modelling, and peptide-fingerprinting mass spectrometry. We show that the S-layer is formed by an uncharacterised 292-kDa S-layer protein (SLP) extending 37 nm from the cytoplasmic membrane, making it, to our knowledge, the largest SLP structurally characterised to date. This SLP has a remarkable multidomain architecture comprising 19 immunoglobulin-like domains, 14 canonical and five non-canonical, organised into a lattice-forming core, a stalk, and a unique crown domain that stabilise the S-layer. Comparative genomic analyses unearthed homologous colossal SLP candidates across Thermoproteota, indicating that this distinctive architecture is conserved across diverse archaeal lineages. Together, our findings provide a structural framework for understanding the cell-surface organisation in hyperthermophilic archaea and suggest that these colossal S-layers represent a specialised adaptation to life at high temperatures.

Uncoupling protein 1 (UCP1) mediates thermogenesis in brown adipose tissue. Whether human-UCP1 shares the bioenergetic properties established for rodent UCP1 (innate uncoupling, GDP sensitivity, fatty acid (re)activation) is not known. Therefore, we expressed human and mouse UCP1 in mouse liver, using adeno-associated viral vectors, and characterized their properties in isolated liver mitochondria. Both UCP1s induced marked innate uncoupling, characterized by increased substrate-supported respiration and decreased membrane potential, in the absence of exogenous fatty acids. Mouse-UCP1 in liver retained the classical regulatory properties of native brown-fat UCP1, including potent inhibition by GDP and reactivation by oleate. In contrast, human-UCP1 was only weakly inhibited by GDP but was strongly responsive to fatty acids. However, ATP potently inhibited human-UCP1, with an apparent IC of {approx}0.4 mM compared with {approx}1.4 mM for GDP, and ATP markedly decreased the sensitivity of human-UCP1 to oleate (re)activation. Despite substantial UCP1-mediated uncoupling, oxidative phosphorylation capacity and mitochondrial OXPHOS protein levels were preserved. Molecular dynamics simulations suggested a structural basis for the species difference. GDP formed persistent interactions with F88 in mouse-UCP1, an interaction absent at the corresponding S88 residue in human-UCP1. In-silico substitution of F88 by serine reduced GDP interaction at this site. Thus, human and mouse UCP1 share innate thermogenic activity but differ fundamentally in nucleotide regulation. The F88/S88 difference may contribute to the preferential GDP sensitivity of mouse-UCP1, whereas ATP provides effective nucleotide control of human-UCP1.

THAP9 is a transposable element-derived gene which encodes a protein that is homologous to the active Drosophila P-element transposase (DmTNP). Both THAP9 and DmTNP possess a C-terminal domain (CTD) which is functionally uncharacterized. Sequence and structural analysis suggest that the THAP9-CTD has a novel fold which is only found in THAP9 homologs. To explore the evolutionary history and characteristics of this novel domain, exhaustive phylogenetic analysis (using MSA, structure prediction, MSTA-based clustering) was performed. THAP9-CTD homologs were more widely distributed throughout the animal kingdom in comparison to DmTNP-CTD homologs which were restricted to arthropods. Moreover, the THAP9-CTD homologs were more conserved, especially among mammals and birds and their average length increased in a class-specific manner. Comparison with the DmTNP-CTD homologs demonstrates that although their respective CTDs may have evolved independently, they both surprisingly share similar secondary structure elements consisting of three conserved helical regions made of hydrophobic residues that are predicted to make up a conserved core. The role of the respective CTDs were further investigated by creating truncation mutants lacking the CTD. Interestingly both THAP9 and DmTNP truncation mutants are still capable of DNA excision and integration suggesting that their respective CTDs are not essential for DNA transposition. Moreover, CTD truncation favours DNA integration in THAP9: this suggests that CTD acquisition during evolution may have led to THAP9 domestication as observed in other transposable element-derived genes like Rag1 and piggybac, which have similar terminal regulatory domains.

Most eukaryotic mRNAs encode a single functional polypeptide. Following translation termination, both the large and small ribosomal subunits are typically released from the mRNA by ribosome recycling factors. However, after translating short upstream open reading frames (uORFs) within the 5 untranslated regions (UTRs), ribosomes can remain associated with the mRNA and reinitiate translation. This process is regulated by the heterodimer MCTS1*DENR (Tma20p*Tma22p in yeast). DENR/Tma22p harbors a SUI1 domain, structurally homologous to the translation initiation factor eIF1/Sui1p, which features a conserved, positively charged {beta}-hairpin loop critical for eIF1 function. Despite this structural similarity, the functional significance of specific elements within DENR/Tma22p remains unexplored. Here, we used in vivo reporter assays in Saccharomyces cerevisiae to quantify reinitiation efficiency following translation of either a short uORF (in the 5 UTR) or a full-length coding sequence (in the 3 UTR). Systematic analysis of single, double, and triple deletions of TMA20, TMA22, and TMA64 (a homolog of Tma20p*Tma22p) revealed that the Tma20p*Tma22p complex exerts a dominant role over Tma64p in modulating reinitiation, while exhibiting functional interplay between the two factors. Using knockout strains complemented with Tma22p variants, we further demonstrated that the positively charged residues of the {beta}-hairpin loop 1 are essential for Tma22p recycling activity. Unexpectedly, deletion of the entire SUI1 domain was less deleterious, and eIF1/Sui1p was able to partially substitute for the SUI1 domain of Tma22p within a chimeric protein context. Our findings establish the {beta}-hairpin loop 1 of the DENR/Tma22p SUI1 domain as a critical determinant for ribosome recycling and reinitiation, and raise the question of whether MCTS1/Tma20p can promiscuously operate with both DENR/Tma22p and eIF1/Sui1p - two specialized factors that evolved from a common structural scaffold to govern distinct steps in the translation cycle.

Eukaryotic translation initiation is tightly regulated by interactions among translation initiation factors (eIFs) that ensure accurate start codon selection. The translation regulator, eIF5 mimic protein 1 (5MP1) contributes to this process by competing with eIF5 for binding to eIF2, thereby increasing the stringency of translation initiation. Despite its important regulatory role and emerging involvement in tumorigenesis, structural information on human 5MP1 remains limited. Here, we report the near-complete backbone and partial side-chain NMR resonance assignments of the C-terminal domain of human 5MP1 (residues 250-419), carrying a W404E substitution that disrupts dimerization. The WT protein forms a dimer at NMR concentrations, which increases the effective size of the protein and also causes disappearance of peaks corresponding to aminoacids at the dimer interface due to conformational exchange. Backbone resonance assignments were completed for 96.4% of the non-proline residues. Secondary structure was analyzed using Chemical Shift Index (CSI) and compared with the AlphaFold structural model. Regions of disagreement between the experimental and computational secondary structure assignments were further examined using 15N-NOESY-HSQC spectra, allowing experimental validation of local structural features. While the AlphaFold model accurately reproduces the overall fold of the 5MP1 C-terminal domain, several localized discrepancies were identified, particularly near the N- and C-terminal regions of the domain, where experimental NMR data support alternative secondary structure assignments. These resonance assignments and experimentally validated structural features provide a foundation for future investigations of the molecular interactions, dynamics, and functions of 5MP1 in translation initiation.

Small annotated open reading frames are the most neglected part of the functionally uncharacterised M. tuberculosis genome. We revisit Rv0810c, a 60-residue protein carrying the unknown-function domain DUF3073 (Pfam PF11273), flagged as an Actinobacteria-signature protein in 2006 but never studied since. Rv0810c is genuinely translated (detected in 11 of 16 M. tuberculosis proteomic datasets), with no significant human homologue, no neighbouring-gene overlap, and no CRISPR-interference polar effect on either flank. Residue-resolved confidence reveals a bipartite architecture: a rigid 33-residue module (pLDDT 91.9) followed by an extended, acidic, intrinsically disordered tail (radius of gyration 22.8 A against 11-12 A expected for a globular protein). The gene is under strong purifying selection (non-synonymous/synonymous ratio 0.86 against 1.93 among 74 size-matched controls, p=5.8x10-8), and its two commonest missense variants are each confined to one sub-lineage, indicating clonal expansion rather than relaxed constraint. DUF3073 is present without a single confirmed loss across 260 well-supported Actinomycetia genera. Despite this conservation, eight independent computational strategies, spanning sequence, structure, electrostatic-patch, embedding-similarity and homo-oligomerisation searches, converge on the same negative: no assignable fold, binding site, or functional neighbour in curated or uncurated sequence space. A phosphosite (Thr24), reproducibly reported by three laboratories, cannot be attributed to a kinase by chemical-genetic or sequence-motif evidence. The contradiction between predicted cytoplasmic topology and macrophage-secretory-fraction detection is narrowed, not resolved: ESX secretion, an immunodominant-epitope confound and host-induced transcription are excluded. Rv0810c exemplifies a class of genuinely uncharacterisable small proteins for which negative reporting, not a manufactured function, is the honest outcome.

Current approaches for imaging fibrotic remodeling have sensitivity, specificity and cost drawbacks that limit both preclinical research and clinical diagnosis. Here, we show that fast green FCF, a small molecule that binds to fibrillar collagen, enables highly sensitive and specific imaging of fibrosis in lung samples from mice and humans using fluorescence microscopy. We report strategies for using fast green FCF staining to assess fibrotic remodeling using precision-cut lung slice and whole-biopsy preparations. Our findings demonstrate that fluorescence imaging of fast green FCF-stained collagen will be useful for fibrosis research and may help to improve detection of fibrosis in clinical pathology.

Mutations in p53 and its 12 isoforms can alter its functions. As N-terminally truncated isoforms of p53 (delta40p53, delta133p53, and delta160p53) participate in tetramer formation, they are important regulators of cancer fate. Although delta40p53- and delta133p53-mediated regulation of cancer is well reported, the mechanism underlying delta160p53 production and its functional role remains unclear. We investigated the internal ribosomal entry site (IRES)-mediated translation of {Delta}160p53 and its role in cancer regulation. As differential synthesis of delta160p53 was observed under different stress conditions, IRES-mediated translation of this isoform was demonstrated using bicistronic luciferase constructs. No cryptic promoters or splicing sites were detected in the IRES sequence. Cell death and late apoptosis were significantly decreased, while proliferation, the number of cells in the S phase, and drug resistance were induced by delta160p53. Furthermore, delta160p53 did not induce p53-responsive promoters. RNA sequencing analysis of delta160p53 overexpression showed similar results, along with the inhibition of other tumor suppressor genes. Overall, our results provide insights into IRES-mediated translation of delta160p53, which can be considered a novel target for cancer treatment.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression primarily in the cytoplasm. However, emerging evidence highlights their additional roles in the nucleus. In particular, spliceosomal miRNAs have been implicated in novel regulatory functions, including the modulation of gene expression. Here, we investigate the nuclear role of spliceosomal miR-99b in breast cancer cells, focusing on its interaction with the long non-coding RNA (lncRNA) SPACA6-AS1. Using non-tumorigenic (MCF-10A) and breast cancer cell lines (MCF-7 and MDA-MB-231), we demonstrate that spliceosomal miR-99b expression increases with malignancy and correlates with elevated SPACA6-AS1 pre-mRNA levels. Notably, miR-99b exhibits full complementarity to the 5-prime splice junction of SPACA6-AS1, suggesting a direct role in splicing regulation. Functional assays reveal that inhibition of miR-99b reduces SPACA6-AS1 pre-mRNA levels, whereas its overexpression enhances pre-mRNA accumulation, indicating that miR-99b promotes the formation or stabilization of the unspliced transcript. Furthermore, increased miR-99b expression is associated with altered ratios of SPACA6 isoforms, supporting a broader role in RNA-level regulation of gene expression. Phenotypically, miR-99b enhances breast cancer cell migration and is required for efficient invasion, particularly in highly aggressive cancerous cells. Our findings uncover a novel nuclear function of miR-99b in modulating lncRNA splicing and gene expression. This spliceosomal miR-99b-SPACA6-AS1 axis represents a previously unrecognized regulatory pathway that contributes to breast cancer progression and may provide a potential target for diagnostic and therapeutic strategies.

Background: Comparisons between independently predicted wild-type and missense-variant protein structures can generate mechanistic hypotheses, but small apparent differences may reflect model-selection variability rather than mutation-specific effects. Methods: Human mitochondrial DNA polymerase gamma (POLG; UniProt P54098) variants p.Arg627Gln (R627Q) and p.Trp748Ser (W748S) were evaluated using five AlphaFold2-PTM network-model outputs per condition generated with one random seed under matched ColabFold settings. Ten pairwise wild type comparisons at each site described between-network model-selection variability. Variant effects were summarized across five within-network wild-type-versus-variant comparisons using rotation-invariant local C-alpha pair distances and local displacement after global and local alignment. Because these comparison designs differ, the wild-type distribution was used as context rather than a mutation-effect null. Wild-type cryo-EM structure 9GGF was used for contact and interface mapping. Experimental A467T and G848S structures 9GGE and 9GGC provided contextual benchmarks. Results: R627Q measurements fell within the range of between-network wild-type differences: its median mean local pair-distance change was 0.170 angstrom, compared with a wild-type median of 0.170 angstrom, and its locally aligned displacement was 0.265 versus 0.248 angstrom. W748S showed higher median values (0.168 versus 0.132 angstrom for pair-distance change; 0.236 versus 0.182 angstrom for locally aligned displacement), but the ranges overlapped and the comparison-design asymmetry precluded a calibrated mutation-effect percentile. Experimental A467T and G848S comparisons produced local changes of similar magnitude. In 9GGF, R627 and W748 directly shared a local microenvironment, with a minimum heavy-atom distance of 3.53 angstrom. R627 also formed short polar-contact candidates with D629 and D743, whereas W748 occupied a hydrophobic packing environment containing Y622 and F750. Both sites were more than 18 angstrom from nucleic acid, more than 30 angstrom from POLG2, and more than 33 angstrom from PZL-A in a ligand-bound structure. Conclusions: Available AlphaFold2 comparisons do not establish a mutation-specific structural deformation for either variant. Experimental-structure mapping supports testable physicochemical hypotheses involving a shared R627-W748 microenvironment - loss of an arginine-centered polar network for R627Q and disruption of a buried aromatic environment for W748S - but not direct DNA, POLG2, or PZL-A contact mechanisms. Matched control substitutions and independent seeds are required to calibrate small mutation-associated structural deltas.

Nitrite can be reduced to nitric oxide (NO) by several heme- and molybdenum-containing proteins, including mitochondrial cytochrome c oxidase (Cco). This activity, designated Cco/NO, has been implicated in hypoxic signaling, but its regulation and quantitative significance relative to other NO-producing systems remain uncertain. We examined its modulation by adenine nucleotides using detergent-solubilized yeast and mouse brain mitochondria supplied with 1 mM nitrite and an ascorbate/TMPD/cytochrome c electron-donor system. ADP and ATP differentially modulated Cco/NO activity, and ADP extended measurable NO formation across the entire oxygen range tested, up to the assay ceiling of 175 {micro}M O2. Nucleotide regulation was also isoform-dependent: ATP slightly inhibited Va-containing Cco but strongly stimulated Vb-containing Cco under anoxic conditions. Rates normalized to cytochrome aa demonstrate multi-turnover nitrite-reductase capacity under these substrate-driven assay conditions. Both the cellular ADP/ATP ratio and subsequently assayed Cco/NO activity increased transiently following a hypoxic shift. These findings establish metabolic and isoform-dependent gating of the catalytic capacity of Cco/NO; they do not establish its fractional contribution to total cellular NO or its operation at physiological nitrite concentrations in intact, coupled mitochondria. This research was supported by CONICET Grant PIP 706 (research team member P.R.C.) and National Institutes of Health Grant GM30228 to R.O.P.

Chlorophyll (Chl) is essential for oxygenic photosynthesis, binding to membrane proteins for light harvesting and electron transfer. In angiosperms, Chl synthesis is halted in darkness, preventing accumulation of Chl-binding photosynthetic complexes in etioplasts. However, etioplasts assemble a dimeric Cytochrome b6f (Cyt b6f) complex, uniquely binding protochlorophyll (Pchl), the esterified derivative of protochlorophyllide (Pchlide). This indicates an evolutionarily conserved structural or functional role for Pchl distinct from the Chl bound in Cyt b6f in chloroplasts. Here we show that upon light-induced Chl synthesis in-vivo and in-vitro, Chl accumulation in Cyt b6f dimers precedes photosystems I and II. We find that chlorophyllide and Chl bind to the light-harvesting-like protein 3 (LIL3), supporting a role for LIL3 in early Chl allocation that extends its described role in stabilizing geranylgeranyl reductase. We determine a dissociation constant of 246.6 {+/-} 37 nM for Chlide binding to LIL3 in-vitro and show that Cyt b6f monomers and LIL3 co-migrate with Chlide in native PAGE, whereas Cyt b6f dimers and LIL3 co-migrate with Chl. These results indicate that Chlide binding to LIL3 chaperones esterification to Chl and reduction of geranylgeraniol, and that Chl release with Cyt b6f dimerization prioritizes Chl binding to Cyt b6f assembly during de-etiolation.

Despite the well-known health risks of neutron exposures, key gaps remain in understanding neutron-induced molecular responses and identifying reliable biodosimetric markers that distinguish neutrons from photon exposure. We provide the first genome-wide analysis of the human blood transcriptional response to an accelerator-derived fission-like spectrum of neutrons versus photons, evaluating transcriptomic relative biological effectiveness (RBE) and radiation quality-discriminating gene signatures. Whole blood from healthy donors was irradiated ex vivo with X-rays (140 kV, 0-4 Gy, n = 3) or neutrons (0.1-8 MeV, 0-1 Gy, n = 2), incubated for 6 h or 24 h, and processed for RNA sequencing from peripheral blood mononuclear cells (PBMCs). Neutrons were markedly more potent than X-rays at inducing differentially expressed genes (DEGs) at equal doses, showing a peak response 6 h post-irradiation followed by a decline. In contrast, X-rays caused a continuous increase in DEGs up to 24 h (neutrons vs. X-rays at 1 Gy: 1,449 vs. 121 DEGs at 6 h; 996 vs. 621 DEGs at 24 h). A universal p53-centered 34-gene signature, including FDXR, EDA2R, GADD45A, and ZMAT3, showed highly monotonic dose responses (Spearman correlation coefficient {approx} 1) across donors, radiation qualities, and timepoints. Additionally, difference-in-differences analysis identified radiation quality-discriminating genes only at 6 h, with transcriptional convergence observed by 24 h, suggesting a very narrow time window for biodosimetric differentiation. We identified a neutron-specific gene signature driven by cGAS-STING-NF-{kappa}B signaling (RELB, NFKB1, C3, MALAT1) and suppression of B-cell and myeloid identity genes (IGHD, TCL1A, CLEC7A, TLR2), defining a biologically coherent neutron quality index with distinct immunomodulatory effects. For the first time, we assessed neutron RBEs at the gene, pathway, and global transcriptomic levels in a human blood model, reporting a global transcriptomic neutron RBE of 1.30 (95% CI: 1.14-1.49) at 6 h and 1.21 (95% CI: 1.14-1.28) at 24 h, providing a valuable basis for biodosimetry in mixed-field exposure scenarios. Our findings advance the mechanistic understanding of neutron radiation responses and support the development of biodosimetric approaches for mixed-field exposure scenarios.

A cnidarian photosymbiont alga, Breviolum minutum, is an emerging model to study symbiosis due its ability to colonise host in absence of light, and amenability to genetic and physiological manipulations. This alga undergoes subcellular reorganisation in response to stress conditions such as elevated temperature and nutrient deprivation. However, subcellular visualisation of this alga is challenging because of its broad spectrum autofluorescence (400-700 nm) and relatively small size (6-8 m). We developed a super resolution imaging, Expansion Microscopy (ExM) workflow - a hydrogel-based technique for mechanical enlargement of cells, that reveals previously inaccessible subcellular features in B. minutum. This ExM workflow presents a set of thermic and enzymatic conditions which enables 4-fold expansion of B. minutum, optical clearing of autofluorescence as well as the removal of its thick cellulose rich cell wall. We implemented a recently described platinum (II)-based tri-functional linker 1, to retain in situ hybridised oligonucleotides targeted to 18S rRNA within ExM hydrogel and exploited its azide reactive group for post-ExM fluorophore labelling (DBCO modification). We observed actin patches (a cytoskeletal feature) and calmodulin (a calcium binding signalling protein) that are not previously visualised in B. minutum. This approach overcomes some of the long-standing problems in visualisation of B. minutum using commonly available reagents and commercially available low-cost ExM compatible chemistries. The broader uptake of this tool for the visualisation of diverse species of photosymbionts will pave the way for fundamental discoveries underpinning cellular reorganisation in formation and breakdown of symbiosis.

The cluster of differentiation 36 (CD36) is a membrane protein with broad physiological roles in health and disease, and its function is regulated in part by phosphorylation. Experimental evidence shows that phosphorylation of Thr92 reduces CD36 affinity for thrombospondin-1 (TSP-1), binding of which initiates antiangiogenic signaling, whereas phosphorylation of Ser237 decreases CD36-mediated fatty acid uptake, with implications for energy metabolism. However, the only available crystal structure of CD36 lacks phosphorylation, and the molecular mechanisms by which phosphorylation regulates CD36 function remain largely unknown. This study provides an atomically detailed computational characterization of CD36 in unphosphorylated and dual phosphorylated states, using molecular dynamics simulations with a total sampling time of 30 microseconds in combination with Markov state models. We present, to our knowledge, the first evidence of a cryptic pocket on CD36 surface that is formed by phosphorylation. This cryptic surface pocket and a loop spanning residues 121-131 form a high affinity binding site for TSP-1 derived ligands, shifting their binding away from the canonical site. We propose that this altered binding provides a molecular basis for the disruption of antiangiogenic signaling upon CD36 phosphorylation. Additionally, our data indicate that, phosphorylation increases helicity and compaction within the helix-loop region spanning residues 296-331, narrowing one of the entrances to the internal cavity and reducing its overall volume. These conformational changes provide a potential mechanistic explanation for the decrease in fatty acid uptake upon CD36 phosphorylation. Our findings provide structural insights that may inform the future design of CD36 modulators and emphasize the importance of targeting phosphorylation induced CD36 conformations in angiogenic and metabolic diseases.